A family of pyrogenic exotoxins, also known as superantigenic toxins, is produced by Staphylococcus aureus and Streptococcus pyogenes. The exotoxins comprised of the S. aureus enterotoxins (SEs) cause the majority of human food poisoning cases manifested by vomiting and diarrhea after ingestion [Schlievert, J Infect Dis 167:997 (1993)]. S. aureus is found widespread in nature, often in association with humans. Among the 5 major serological types within the family of SEs (labeled SEA to SEE and SEG), SEB is the most prominent [Marrack and Kappler, Science 248:705 (1990)]. SEB has also been recognized as a leading cause of human cases of non-menstrual toxic shock syndrome that can accompany surgical or injurious wound infections, as well as viral infections of the respiratory tract of influenza patients to which children are especially vulnerable [Schlievert (1993) ibid.; Tseng et al., Infect Immun 63:2880 (1995)]. Toxic shock syndrome, in its most severe form, causes shock and death [Murray et al., ASM News 61:229 (1995); Schlievert (1993) ibid.]. More generally, members of the staphylococcal exotoxin family, including SEA to SEE and toxic shock syndrome toxin 1 (TSST-1), have been implicated in toxic shock syndrome, in atopic dermatitis [Schlievert (1993) ibid.] and in Kawasaki's syndrome [Bohach et al., Crit Rev Microbiol 17:251 (1990)].
Because of the potential for causing lethal shock in humans after aerosol exposure and because of the relative ease with which SEB can be produced in large amounts, there is concern that SEB could be used as a biological weapon [Lowell et al., Infect Immun 64:1706 (1996)]. SEB is thought to be a potential biological weapon mainly in view of its lethal potential. However, through its exquisite ability to induce vomiting and diarrhea, SEB is also an incapacitating agent that could severely impair the effectiveness of a fighting force, even temporarily, thereby enhancing vulnerability to conventional military means. Needless to say, the harmful effects of SEB need to be generally attacked, and not only in connection with the military aspect.
SEB is a toxic mitogen that triggers a paradoxical response in the infected organism: a vast stimulation of the immune system on the one hand and, on the other hand, a profound immunosuppression that may allow the multiplication of the infecting bacteria, unimpeded by an immune response [Hoffman, Science 248:685 (1990); Smith and Johnson J Immunol 115:575 (1975); Marrack et al., J Exp Med 171:455 (1990); Pinto et al., Transplantation 25:320 (1978)]. During the cellular immune response, a dynamic interplay is induced, by antigens or mitogens, between activation of Th1 type cytokine gene expression, exemplified by interleukin-2 (IL-2), interferon-γ (IFN-γ and tumor necrosis factor-β (TNF-β), and on the other hand, its cell-mediated suppression by CD8 cells and other cell subsets [Ketzinel et al., Scand J Immunol 33:593 (1991); Arad et al., Cell Immunol 160:240 (1995)], and by the inhibitory cytokines from Th2 cells, IL-4 and IL-10 [Mosmann and Coffman, Annu Rev Immunol 7:145 (1989)].
SEB is a member of the family of pyrogenic exotoxins [Herman et al., Ann Rev Immunol 9:745 (1991)] that comprises bacterial exotoxins and Mls proteins. These antigenic toxins stimulate a 20,000-fold greater proportion of rodent or human T cells than do ordinary antigens. Thus, SEB activates 30–40% of all T cells in some mice to divide and produce cytokines [Marrack and Kappler (1990) ibid.]. Indeed, toxicity of SEB requires T cells; mice that lack T cells or SEB-reactive T cells are not affected by doses of SEB that cause weight loss and death in normal animals [Marrack et al. (1990) ibid.; Marrack and Kappler (1990) ibid.]. Unlike normal antigens, SEB and related toxic mitogens do not require processing and antigen presentation [Janeway et al., Immunol Rev 107:61 (1989)] but activate the T cell by binding at a specific site in the variable portion of the β chain (V-β) of the T-cell receptor [Choi et al., Nature 346:471 (1990)]. The crucial region for T-cell receptor interaction with toxin lies on the outer face of the V-β domain, a region not involved in conventional antigen recognition [Choi et al., Proc Natl Acad Sci U.S.A. 86:8941 (1989)]. Simultaneously, pyrogenic exotoxins bind directly to MHC class II molecules [Scholl et al., Proc Natl Acad Sci U.S.A. 86:4210 (1989)] and thus affect primarily CD4+ T cells, although CD8+ cells are also activated [Fleischer and Schrezenmeier, J Exp Med 167:1697 (1988); Fraser, Nature 339:221 (1989); Misfeldt, Infect Immun 58:2409 (1990)]. The current consensus is that pyrogenic exotoxins activate T cells so effectively because they bypass the ordinary interaction of antigen with class II MHC and T-cell receptor [Janeway, Cell 63:659 (1990)]. An alternative view is that pyrogenic exotoxins act as coligands that facilitate, and thus greatly exaggerate, the effect of minute amounts of ordinary antigens [Janeway (1990) ibid.].
The toxicity of SEB and related exotoxins is thought to be related to the capacity of these molecules to stimulate the rapid and excessive production of cytokines, especially of IL-2, IFN-γ and tumor necrosis factors (TNFs). IL-2, IFN-γ, and TNF-β are secreted from activated T helper type 1 (Th1) cells while TNF-β is secreted by Th1 cells, monocytes and macrophages. High levels of these cytokines, suddenly produced, have been implicated as a central pathogenic factor in toxin-related toxicity [Schad et al., EMBO J 14:3292 (1995)] and are thought to cause a rapid drop in blood pressure leading to toxic shock.
While investigation has produced a plausible explanation for the vast stimulation of T cells by SEs, it is not yet clear why these toxins are also strongly immunosuppressive. They induce a decline in both primary T and B cell responses, including the production of antibodies and the generation of plaque-forming cells [Hoffman (1990) ibid.; Smith and Johnson (1975) ibid.; Marrack et al. (1990) ibid.; Pinto et al. (1978) ibid.; Ikejima et al., J Clin Invest 73:1312 (1984); Poindexter and Schlievert, J Infect Dis 153:772 (1986)].
The sensitivity of humans to staphylococcal toxins exceeds that of mice by a factor of 100. Thus, the toxic shock syndrome toxin 1, TSST-1, another pyrogenic exotoxin from Staphylococcus aureus, stimulates human T cells to express the key cytokines, IL-2, IFN-γ and TNF-β at <0.1 pg/ml, while murine cells require approximately 10 pg/ml [Uchiyama et al., J Immunol 143:3173 (1989)]. Mice may have developed relative resistance to toxic mitogens by deleting from their T cell repertoire those cells that display the most highly reactive V-β chains or by eliminating these V-β genes [Marrack and Kappler (1990) ibid.]. Such deletions have not been detected in humans, making them far more vulnerable.
The incapacitating and potentially lethal effects of SEB in humans (and of exotoxins of the same family of superantigens), whether exerted on civilians or military personnel, create a need for prophylaxis against SEB, for treatment of SEB-exposed individuals and for a safe SEB vaccine.
Despite the urgency of this need, methods of protection or treatment have been lacking. Thus, in D-galactosamine-sensitized murine models of SEB intoxication, one based on intramuscular challenge with SEB toxin and the other on intranasal challenge using mucosal SEB exposure, it was possible to protect mice with proteosome-SEB toxoid vaccines in which the SEB toxoid component was prepared by a 30-day formalin treatment of the biologically active, intact SEB protein molecule [Lowell et al. (1996) ibid.]. As detailed below, however, the inventors have now found that antibodies raised against certain peptide domains within the SEB molecule enhance the ability of SEB to stimulate human T cells, rather than protecting them against the toxin. This finding limits the use of SEB toxoids as vaccine, in view of the danger of eliciting certain SEB-sensitizing antibodies that could not only fail to confer protective immunity but would lead to significant exacerbation of the toxic responses in SEB-exposed persons.
Other investigators sought recourse in the use of fragments rather than the complete SEB protein molecule, through the synthesis of a series of overlapping SEB peptides, in the order of 30 amino acids each in length [Jett et al., Infect Immun 62:3408 (1994)]. These peptides were used to generate antisera in rabbits whose ability to inhibit the SEB-induced proliferation of a mixture of human T cells and macrophages was then tested. That effort failed to yield an effective or specific inhibitory response. Thus, peptide pSEB(113–144), containing amino acids 113 to 144 of the SEB protein molecule, as well as peptides covering amino acids 130–160, 151–180, and 171–200 each elicited antisera that inhibited the SEB-induced lymphocyte proliferation weakly, by up to 2.5-fold [Jett et al. (1994) ibid.].
A number of investigators attempted to create peptide vaccines. Thus, Mayordomo et al. [J Exp Med 183:1357 (1996)] used a mutant peptide derived from p53 as vaccine for therapy of murine tumors. Hughes and Gilleland [Vaccine 13:1750 (1995)] used synthetic peptides representing epitopes of outer membrane protein F of Pseudomonas aeruginosa to afford protection against P. aeruginosa infection in a murine acute pneumonia model. In an attempt to use peptide immunization in humans Brander et al. [Clin Exp Immunol 105:18 (1996)] showed that a combined CD8+/CD4+ T cell-targeted vaccine restimulated the memory CD4+ T cell response but failed to induce cytotoxic T lymphocytes.
Major sources of exotoxins are, as already mentioned, S. Aureus and S. Pyogenes. The flesh-eating bacteria, S. Pyogenes, produce a family of different toxins with closely similar mode of action: excessive activation of T cells. S. Aureus produces next to SEB as major component, also SEA, SECs, SEE and TSST-1 (toxic shock syndrome toxin 1) and S. Pyogenes produces SPE A as major toxin, as well as other pyrogenic exotoxins. Hence, in staphylococcal food poisonings and, more seriously, in biological warfare or in toxic shock caused by S. pyogenes, mixtures of toxins are encountered. The composition of such mixtures cannot be anticipated with certainty. The worst scenarios of biological warfare entail not the use of a single, purified pyrogenic exotoxin, as favored for immunological studies, but rather readily attainable, crude natural mixtures of such toxins, as produced, for example, by culturing S. Aureus. 
Clearly, this complexity demands the development of broad-spectrum antagonists of pyrogenic exotoxins as well as broad-spectrum vaccines.
There exists, therefore, a long-felt need to design a SEB vaccine that is free of sensitizing potential, yet is capable of protecting test animals or humans against lethal doses of toxin. Even greater value would be a vaccine that can afford protection not only against SEB, but also against a wider spectrum of the SE toxin family, including, for example, SEA, SEC, TSST-1, etc.
Moreover, currently, there is no prophylaxis available against SEB or any other pyrogenic exotoxin, nor treatment of exposed persons. There exists, therefore, also a long-felt need to design agents that antagonize the action of SEB, as well as any other pyrogenic exotoxin. Such antidotes will have great value, both in the medical treatment of acute food poisoning and in saving lives in cases of toxic shock and related pathological conditions.
There exists therefore a need for an antagonist against pyrogenic exotoxins, for use in immediate treatment, or short term prevention and rapid prophylaxis, of acute toxic shock and of the harmful effects of such toxins which may be due to, for example, accidental food poisoning, and for a vaccine for immunization against intoxication by pyrogenic exotoxins, for long term protection thereagainst.
In addition, currently there is no way by which to assess the efficacy of vaccination of humans against pyrogenic toxins, since humans cannot be challenged with the toxin in order to check whether they have been conferred the desired immunity. There exists therefore a need for a clinical test for assessing the efficacy of vaccination of humans against pyrogenic toxins which employs surrogate markers.
The inventors have designed a series of peptide antagonists, in particular a 12-mer peptide and a 14-mer peptide, which block the action of pyrogenic exotoxins on the human immune response in vitro, severely inhibiting toxin-mediated induction of IL-2, IFN-γ and TNF-β mRNA. It is clear that these peptides could be used for treatment of acute toxic shock and of harmful effects which may be due to, for example, accidental food poisoning induced by pyrogenic exotoxins. In addition, antibodies raised against individual peptides protect against many pyrogenic exotoxin-mediated responses, suggesting the potential development as broad-spectrum vaccines.